Although polyomaviruses (PyV) typically cause lifelong and asymptomatic infections in healthy humans, they can cause severe illness in immunocompromised or elderly patients. The discovery of Merkel cell polyomavirus (MCV) in Merkel cell carcinomas (MCC) has refocused the significant role of PyV in human disease. Cellular transformation and oncogenesis by PyV is dependent on large T (LT) and small T (ST) antigens binding to cellular proteins. The PyV T antigens induce cell growth control by binding to key host factors and inducing significant perturbations in important cellular networks. For example, the SV40 ST contributes to cellular transformation by binding to the A and C subunits of protein phosphatase PP2A while displacing the B subunit of PP2A. It was recently shown that MCV-ST could transform rat fibroblasts in a PP2A-independent manner. Notably, a large-scale and systematic proteomic screen of viral proteins in normal human cells revealed a highly enriched and novel interaction between MCV-ST and the nuclear receptor SET domain-containing protein 1 (NSD1). NSD1 activates NF-kB through direct methylation; constitutive activation of NF-kB is a hallmark in most cancers. This application is focused on determining whether the transforming ability of MCV-ST depends on host cell factors, in addition to PP2A binding, including NSD1. This application will test the hypothesis that MCV and SV40 ST can contribute to cellular transformation in a PP2A independent manner. To test the hypothesis, the following 3 Specific Aims will be performed: (SA1) Determine if the unique domain of MCV-ST and SV40-ST contributes to cellular transformation in a PP2A- independent manner. I will compare the transforming activity of SV40-ST with MCV-ST in BJ-hTERT-RasV12- SV40LT cells, which become transformed with the addition of SV40-ST. I will determine if SV40 and MCV ST mutants that fail to bind to PP2A but leave other properties intact can induce cellular transformation. I will test the transforming activity of chimeras that swap the N-terminal J and unique domains of SV40-ST and MCV-ST. (SA2) Determine if MCV-ST interacts with NSD1 in a PP2A-independent manner and if this interaction contributes to MCV-ST mediated transformation. Validation of this interaction will be pursued in vitro using various MCV-ST constructs in normal and MCC cell lines. I will determine the specific residues of MCV-ST that are required for binding to NSD1, and if this interaction is dependent on PP2A binding. RNAi knockdown of NSD1 will be performed to determine the requirement for NSD1 in MCV-ST-mediated transformation. The histone methyltransferase activity of NSD1 will be assayed in vitro and in complex with MCV-ST. (SA3) Determine if MCV-ST and NSD1 have an effect on NF-kB activation. The potential impact of MCV-ST-NSD1 complex formation on activation of NF-kB, a direct target of NSD1, will be evaluated. If successful, this application will provide evidence tha PyV ST can transform cells in a PP2A dependent and independent manner.